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Am J Physiol Heart Circ Physiol 287: H567-H572, 2004. First published April 1, 2004; doi:10.1152/ajpheart.01145.2003
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Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet

Diego López,1 Xavier Orta,2 Kelly Casós,1 M. Puy Sáiz,1 Pere Puig-Parellada,3 Mireia Farriol,2 and M. Teresa Mitjavila1

1Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, E-08028 Barcelona; 3Unitat de Farmacologia, Facultat de Medicina, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, E-08026 Barcelona; and 2Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d'Hebron, E-08035 Barcelona, Spain

Submitted 22 December 2003 ; accepted in final form 24 March 2004


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (·NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the ·NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular ·NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, L-arginine, and L-citrulline were also evaluated in homogenates. Superoxide anion (O2–·) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%); ·NO production increased by 90%, cGMP levels increased by 100%, and L-arginine decreased by 30%. No change in aortic O2–· production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.

polyunsaturated fatty acids; endothelium; nitric oxide; superoxide anion; free radicals

FISH AND FISH OILS are the main source of human dietary long-chain {omega}-3 polyunsaturated fatty acids. A fish oil-rich diet performs multifaceted actions (8). It replaces arachidonic acid by eicosapentanoic acid (20:5{omega}-3) and docosahexanoic acid (22:6{omega}-3) in phospholipid membranes (34, 36), induces changes in eicosanoid metabolites, and impairs free radical release in different types of stimulated cells (6, 7, 22, 37, 43).

It is widely accepted that damage to the endothelium plays a key role in the development of early atherosclerosis and that fish oils prevent atherosclerosis (10, 14) and significantly improve endothelial function in hypercholesterolemic subjects (44). The accumulation of oxidized lipids in the vascular wall suggests that free radical-mediated tissue injury may be a focal point in the formation of atherosclerotic lesions (12, 23). However, nitric oxide (·NO) has been described as essential for the regulation of vascular tone and hemodynamics (13), and ·NO also displays antiatherogenic properties both in vivo (31) and in vitro (21). We previously found (18) that a fish oil-rich diet increases the acetylcholine-mediated relaxation of rat aortic segments. This increase is due neither to a decrease in the activation of the cyclooxygenase pathway nor to a greater response of smooth muscle to ·NO but is an endothelium-dependent relaxation that involves ·NO release (18). It is likely that the incorporation of {omega}-3 polyunsaturated fatty acids into phospholipids of cell membranes induces changes in eicosanoid metabolites that can modulate the rates of ·NO production by the vessel. On the basis of these considerations, the present study addressed the question of whether the mRNA and/or protein levels of endothelial NO synthase (eNOS), an enzyme that catalyzes ·NO synthesis, leading to smooth muscle relaxation through activation of guanylyl cyclase and generation of cGMP, are affected by a fish oil-rich diet.


   METHODS
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 METHODS
RESULTS
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Diets and aortic preparation. After being weaned, two groups of 14 male Sprague-Dawley rats (Harlan Interfauna Ibérica, Barcelona, Spain) each were fed for 8 wk on semipurified diets containing 100 mg/kg of all rac-{alpha}-tocopherol acetate (equivalent to 60 IU/kg of {alpha}-tocopherol) and 5% lipids prepared in our laboratory (Table 1). The lipids were either corn oil (CO; rich in 18:2{omega}-6) or menhaden oil (MO; rich in 20:5{omega}-3 and 22:6{omega}-3). Diets were prepared weekly and stored at –20°C to prevent oxidation, and food was provided and removed daily. Body weight was recorded every week. At the end of the feeding period, rats were anesthetized with sodium urethane (1.5 g/kg ip) and exsanguinated. Plasma was obtained after centrifugation at 1,770 g for 15 min at 4°C, and an aliquot was frozen at –80°C. The thoracic and abdominal aortas were cut in segments as previously described (18). The procedures and the care of the rats complied with European Community guidelines.


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  		Table 1. Composition of semipurified diets

 
Assays for NOS. Aortic segments (~25 mg) were homogenized in 1 ml of TRIzol reagent to determine eNOS mRNA expression. Total RNA was extracted and quantified by measuring the absorbance at 260 nm. Real-time RT-PCR using a pair of hybridization probes to detect the target and the reference amplicon of the aortic RNA extract was used to quantify eNOS mRNA expression. GAPDH mRNA, the expression of which is independent of dietary fat composition (41), was used as a housekeeping control to correct for sample inhomogeneity. RT-PCR amplification by a Light Cycler instrument (Roche, Basel, Switzerland) was carried out as described previously (29). In each reaction, 150–300 ng RNA from the aorta was added and the MgCl2 concentration was adjusted to 6 mM. The rat eNOS primers were 5'-TTCCGGCTGCCACCTGATCCTAA-3' and 5'-AACATGTGTCCTTGCTCGAGGCA-3'. The rat GAPDH primers were 5-'TCCCTCCAGATTGTCAGCAA-3' and 5'AGATCCACAACGGATACATT-3'. After 30 min at 55°C for transcription, there were 45 rounds of amplification at 90°C (5 s), 64°C (15 s), and 72°C (15 s). Relative quantification was performed by measuring the crossing point (Cp; the cycle at which the fluorescence reached the exponential phase) for each reaction, and the difference between eNOS and GAPDH ({Delta}{Delta}Cp) was calculated and expressed as 2–{Delta}{Delta}Cp.

Aortic segments (~10 mg) were homogenized in 500 µl of a lysis buffer containing detergent to evaluate eNOS protein levels and NOS activity. Homogenate samples (100 µg protein/lane) for eNOS protein levels were submitted to gel electrophoresis and blotted onto a nitrocellulose membrane. Purified bovine eNOS was also loaded (2 µg) as a positive control, and a prestained protein standard was used to check transfer efficiency. Membranes were then exposed to a 1:5,000 dilution of rabbit anti-human eNOS polyclonal antibody for 1 h. Antibody detection was carried out by using an Immun-Star Anti-Rabbit Detection Kit (Bio-Rad, Hercules, CA). For semiquantitative analysis, Western blot images were exported as TIFF files, and for quantification rectangles were set to identify specific bands; intensities were calculated as the integrated intensity of all pixels inside the rectangles, and background correction was applied using values of a selected rectangle. Results are expressed in relative densitometric units relative to bovine eNOS.

Homogenate samples (20–40 µg protein/tube) were used to measure NOS activity by the conversion of L-[3H]arginine to L-[3H]citrulline with a Cayman kit. In some experiments, 1 mM NG-nitro-L-arginine methyl ester (L-NAME) HCl, an inhibitor of NOS, was added to the incubation medium for 30 min. To address the influence of eNOS on NOS activity, the endothelium was removed in some aortic segments by gently rubbing the intima surface with a stainless steel wire. Results are expressed as picomoles per milligram of total protein per hour.

Endothelial ·NO production by electron spin resonance. The detection of ·NO took place according to the Vanin technique (42). Three aortic segments (5–10 mg) from the same rat were stripped and randomized; two of these were left intact, and the third was endothelium denuded. They were preincubated at 37°C for 20 min in Ringer solution (pH 7.4) and then exposed to the spin-trapping agents diethyldithiocarbamic acid (DETC; 5 mM final concentration) and FeSO4·7H2O (50 µM final concentration) for 30 min. Aortic strips were then weighed, frozen in liquid nitrogen, and stored at –80°C for posterior electron spin resonance (ESR) analysis. One of the intact strips was preincubated for 30 min in the presence of 1 mM NG-nitro-L-arginine (L-NNA), an inhibitor of NOS. The ESR-detectable paramagnetic complex was evaluated in a Bruker 300E spectrometer (Bruker Instruments, Billerica, MA). The signal due to the complex corresponded to the difference in intensity between a maximum at 3,440 G and a minimum at 3,470 G. A standard curve of DETC-Fe-·NO was generated by diethylamine NONOate (10 nM–100 µM; linearity interval was between 100 nM and 3 µM). This curve was used to extrapolate the Fe-·NO-DETC signal and also to estimate the linearity of the assay. Results are expressed as relative intensity units per milligram of tissue.

Vascular cGMP content. Aortic segments (~10 mg) were homogenized in 500 µl of Ringer solution for cGMP measurement (2) with an enzyme immunoassay kit (Cayman). Immunoassay showed linearity between 0.05 and 1.5 nM cGMP, and homogenates were diluted to fit this interval. Results are expressed as picomoles per gram of protein.

Measurement of L-arginine and L-citrulline in aortic homogenates. Aortic segments (~10 mg) were used to determine the levels of L-arginine and L-citrulline free amino acids. The amino acid analysis was conducted by an ion-exchange chromatograph (Alpha Plus Two; Pharmacia LKB Biotechnology, Uppsala, Sweden) coupled to an autoanalyzer. The 300-mm-length and 3-mm-width column was filled with Dionex DC 6A (Dionex, Sunnyvale, CA).

Lithium citrate buffers were used as recommended by the manufacturer (Hilger Analytical; Margate, UK). The fluorescent derivatives produced by orthophthaldialdehyde were detected in a fluorimeter (395 nm excitation, 475 nm emission) (Kontron SFM-25; Rotkreuz, Switzerland). Norleucine was used as internal standard. The results are expressed in picomoles per milligram of tissue.

Vascular O2–· production and activities of enzymatic antioxidants. O2–· production was measured in thoracic aortic segments by chemiluminescence according to the technique of Pagano et al. (30) with some modifications (18). Assays were carried out with 5 µM lucigenin in the absence or presence of 60 U/ml SOD or 1 mM NG-monomethyl-L-arginine (L-NMMA). The signal was evaluated with a luminometer (Bio Orbit; Turku, Finland). Results are expressed as arbitrary units of light per square millimeter per minute.

Aortic segments (5–7 mg) with a functional endothelium were homogenized by Polytron (Kinematica, Littau, Switzerland) in 2 ml of phosphate buffer (50 mM, pH 7.0). SOD activity was assayed by the inhibition of pyrogallol autooxidation (19). Results are expressed as units per milligram of tissue (1 unit induces an inhibition of 50% pyrogallol autooxidation). Catalase activity was assayed by H2O2 consumption, measured over 15 s by spectrophotometry following the Aebi technique (1) modified by Pieper et al. (32). The first-order reaction rate (k) of H2O2 consumption by catalase was calculated, and the results are expressed in k per milligram of tissue.

Materials. The following chemicals and reagents were obtained from Sigma (St. Louis, MO): oils, SOD, DETC, and FeSO4·7H2O. The lysis buffer, purified bovine eNOS, eNOS polyclonal antibody, diethylamine NONOate, NOS inhibitors, and NOS activity kit were from Cayman (Ann Arbor, MI). RNA primers and probes were obtained from Roche (Mannheim, Germany).

Statistical analysis. Data are presented as means ± SE. Statistical evaluation was performed by the Student's t-test for unpaired observations.


   RESULTS
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Growth performance. No significant differences in daily food consumption were observed between the two dietary conditions. The increase in body weight over the 8-wk periodwas similar in the two groups of rats; thus there was no significant difference in final body weight (341 ± 9 and 329 ± 5 g for CO- and MO-fed rats, respectively).

Assays for NOS. The eNOS mRNA content in the aorta increased by 31%, as expressed by 2–{Delta}{Delta}Cp (0.0209 ± 0.0010 and 0.0273 ± 0.0006 for CO and MO, respectively), in rats fed the MO-rich diet (Fig. 1).



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  		Fig. 1. Relative endothelial nitric oxide (NO) synthase (eNOS) mRNA expression by quantitative RT-PCR in total aortic RNA from aortic segments (25 mg) from rats fed a corn oil (CO)- or menhaden oil (MO)-rich diet. RT-PCR of GAPDH mRNA was used as a housekeeping control. Individual values and means are shown; n = 7 rats/diet. Cp, crossing point.

 
Representative Western blots conducted on eNOS protein from homogenates of the thoracic aorta from rats fed CO- or MO-rich diets are shown in Fig. 2A. The accompanying histograms (Fig. 2B) illustrate the mean of the eNOS protein levels (relative densitometric units) for each group. The aortic eNOS protein levels were increased (P < 0.001) in MO-fed rats versus CO-fed rats (1.53 ± 0.22 and 0.90 ± 0.10 relative densitometric units, respectively).



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  		Fig. 2. A: representative Western blots of eNOS carried out in aortic homogenate (100 µg protein/lane) with a standard of bovine eNOS (2 µg; lane a), from rats fed the CO-rich diet (lane b), or from rats fed the MO-rich diet (lane c) exposed to a 1:5,000 dilution of rabbit anti-human polyclonal antibody for eNOS. B: eNOS protein levels. Data are means ± SE; n = 4 rats/diet. **P < 0.01.

 
NOS activity (Fig. 3) was significantly higher (P < 0.01) in homogenates of thoracic aorta with a functional endothelium from rats fed the MO diet than in those from rats fed the CO diet (90.0 ± 6.7 and 44.5 ± 3.7 pmol·mg total protein–1·h–1, respectively). L-[3H]citrulline formation by aortic homogenates from both groups of rats was almost completely blocked by the addition of 1 mM L-NAME (3.1 ± 0.2 and 2.5 ± 0.2 pmol·mg total protein–1·h–1 for CO and MO, respectively). NOS activity in endothelium-denuded segments was undetectable, indicating that the observed increase of NOS activity in intact segments is originated from the endothelium.



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  		Fig. 3. NOS activity of aortic homogenates (20–40 µg protein) from rats fed a CO- or MO-rich diet measured by the conversion of L-[3H]arginine to L-[3H]-citrulline. In some experiments, 1 mM NG-nitro-L-arginine methyl ester HCl was added to the incubation medium for 30 min. Data are means ± SE; n = 6 rats/diet. **P < 0.01.

 
Endothelial ·NO production by ESR. An aortic strip with a functional endothelium from rats fed a standard chow incubated in the presence of DETC and FeSO4 exhibited an ESR feature (Fig. 4A) that can be superimposed on the two signals by using 0.1 mM NONOate as a ·NO donor (Fig. 4B). Incubation of the aortic strips with functional endothelium from rats fed the CO and MO diets in the presence of the DETC-Fe complex gave the ESR shown in Fig. 4, C and D, respectively. The appearance of the DETC-Fe-·NO feature was reduced by 1 mM L-NNA (Fig. 4E). The dietary administration of MO for 8 wk significantly increased (90%; P < 0.001) the bioavailability of ·NO in vascular tissue (Fig. 5). Incubation with L-NNA reduced the signal by 92.8 ± 3.7% and 95.1 ± 4.6% in rats fed CO and MO diets, respectively. The endothelium-denuded strips gave a very small signal per milligram of tissue in both dietary groups, indicating that the endothelium was the main source of ·NO.



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  		Fig. 4. Representative electron spin resonance (ESR) spectra of aortic segments (5–10 mg) preincubated at 37°C for 20 min in Ringer solution and then exposed to the spin-trapping agents diethyldithiocarbamic acid (DETC; 5 mM) and FeSO4 (50 µM) for 30 min. The DETC-Fe-·NO complex was evaluated in aortic segments as described in MATERIALS AND METHODS: from a rat fed a standard chow diet (A), after incubation with 100 µM diethylamine NONOate (B), from a rat fed the CO-rich diet(C), from a rat fed the MO-rich diet (D), and from a rat fed the MO-rich diet incubated in the presence of 1 mM NG-nitro-L-arginine (E).

 


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  		Fig. 5. ·NO production by aortic segments from rats fed a CO- or MO-rich diet evaluated by ESR of DETC-Fe-·NO complex. Data are means ± SE; n = 6 rats/diet. ***P < 0.001.

 
Vascular cGMP content. The thoracic aortas from rats fed the MO diet presented more cGMP (Fig. 6) than aortas from rats fed the CO diet (45.4 ± 3.0 vs. 21.4 ± 2.3 pmol/g total protein, respectively; P < 0.001).



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  		Fig. 6. cGMP content in homogenates of aortic segments (~10 mg) from rats fed a CO- or MO-rich diet. Data are means ± SE; n = 6 rats/diet. ***P < 0.001.

 
L-Arginine and L-citrulline in aorta. The L-arginine-to-L-citrulline ratio was lower in aortic homogenates from rats fed the MO-rich diet (Fig. 7A) than in those from rats fed the CO-rich diet (6.4 ± 1.2 vs. 13.1 ± 1.6, respectively). This was due to a decrease in L-arginine content (534 ± 41 vs. 357 ± 41 pmol/mg tissue for CO- and MO-fed rats, respectively; Fig. 7B) and an increase in L-citrulline content (42 ± 3 vs. 60 ± 7 pmol/mg tissue for CO- and MO-fed rats, respectively; Fig. 7C).



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  		Fig. 7. Arginine and citrulline content in homogenates of aortic segments (~10 mg) from rats fed a CO- or MO-rich diet. Amino acids were separated by ion-exchange chromatography and analyzed in an autoanalyzer. A: arginine-to-citrulline ratio. B: arginine content. C: citrulline content. Data are means ± SE; n = 6 rats/diet. *P < 0.05; **P < 0.01.

 
Vascular O2–· production and activities of enzymatic antioxidants. O2–· production by aortic segments was similar in thetwo dietary groups (Fig. 8). Incubation with SOD reduced O2–· by 60% in the two groups of rats. Incubation with L-NMMA significantly increased the levels of O2–· (P < 0.05), indicating that ·NO was produced. SOD and catalase activities (Table 2) were also similar in aortic homogenates from rats fed the CO- and MO-rich diets.



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  		Fig. 8. NADPH-stimulated superoxide anion (O2–·) production by aortic segments (5–7 mg) from rats fed a CO- or MO-rich diet. Assays were performed in the absence (Ctrl) or presence of 60 U/ml SOD or 1 mM NG-monomethyl-L-arginine (L-NMMA). Data are means ± SE; n = 6 rats/diet. *P < 0.05; **P < 0.01.

 

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  		Table 2. SOD and catalase activities in aortic homogenates from rats fed CO- or MO-rich diets

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study provides the first direct evidence that the ingestion of a fish oil-rich diet results in upregulation of eNOS mRNA and protein levels, which induces the subsequent increase of endothelium-dependent vascular relaxation. Various mechanisms of action have been proposed that involve the biological effects of {omega}-3 polyunsaturated fatty acids on the cardiovascular system (antiarrhythmic actions, antithrombotic effects, and plasmatic cholesterol and triacylglycerol reduction; Ref. 8). Our results explain the mechanisms involved in the responses observed in other studies, showing that dietary fish oil increases the endothelium-dependent relaxation induced by vasoactive substances (18, 38, 39) and also the cGMP content in the vascular wall (6). These contributions open a new point of view on the mechanisms of action of fish oils at the blood vessel level. Moreover, the ·NO-cGMP system is a key physiological signaling pathway involved in the regulation of vascular tone and blood pressure (15).

Shimokawa and Vanhoutte (38) postulated that a chronic treatment with polyunsaturated fatty acids mainly affected the plasmatic membrane of the endothelial cells by modulating the sensitivity of the receptor-operator release of endothelium-derived relaxing factor from the endothelium. The same authors also maintained that 20:5{omega}-3, by altering the fluidity of endothelial cell membranes, results in facilitated release of endothelium-derived relaxing factor. Recent studies on cultured endothelial cells (24, 26, 28) indicate that 20:5{omega}-3 increases intracellular Ca2+ levels and the translocation of eNOS protein from the caveolar microdomain to the cytosol. Li et al. (16) demonstrated that activation of protein kinase C enhances the transcription of the human eNOS gene. The very small remaining ·NO signal observed by ESR in endothelium-denuded strips confirms that although the three distinct isoforms of NOS have been characterized in several vascular cell types (3, 4, 20) the endothelium is the main source of ·NO. The constitutively expressed eNOS is an extremely important homeostatic regulator of numerous physiological processes. In the present study we observed increases in eNOS mRNA (31%) and protein (72%) levels. Subsequent increases in endothelial ·NO bioavailability (90%) and aortic cGMP (100%) may be due to the upregulation of eNOS expression and not to an enhancement of NOS activity, as NOS activity values did not change when expressed by eNOS protein (33.6 ± 4.7 and 28.4 ± 3.2 pmol·µg total protein–1·h–1 for MO and CO, respectively). However, the mechanisms underlying the increased expression of eNOS in response to dietary MO in the present study remain unclear, although the main contributor appears to be eNOS expression. The increase in ·NO bioavailability in endothelium-intact aortic segments together with the very small ·NO signal by ESR in endothelium-denuded aortic segments indicates that the MO acts on the endothelium. These findings can explain the previous results of our group (18), which showed a 68% increase in acetylcholine-mediated relaxations in aortic segments from rats fed a MO-rich diet. The present studies performed ex vivo give more convincing evidence than experiments using endothelial cells in culture (5) because the chemical and mechanical stimuli from the systemic vasculature, which also modulate eNOS protein levels, are absent in culture systems.

The increase in ·NO production by dietary fish oils may explain either their preventive effect in the development of cardiovascular disease (10) or the improvement of endothelial functions in hypercholesterolemic subjects (44). This increase in ·NO has several key features at the platelet and vascular level, but we will comment on only two. First, ·NO increases blood vessel relaxation, either in basal (39) or mediator-stimulated (18) production. Second, ·NO can also act as a potent chain-breaking antioxidant preventing low-density lipoprotein oxidation (9, 25, 35) and thus atherogenesis (21). The antioxidant property of ·NO can only be manifested when prooxidative reactions do not predominate as it reacts with O2–· to generate peroxynitrite, a much more reactive species that impairs nitrovasodilator relaxation (27) and increases the susceptibility of low-density lipoprotein to oxidation (11). The relative rates of ·NO and O2–· are critical in determining the outcome of biological oxidation (17, 33). To further ascertain that the MO-rich diet increases the bioavailability of ·NO, we also assessed O2–· generation by aortic segments. The maintenance of the same levels of O2–· production after rats are fed with the MO-rich diet is sustained by the absence of changes in SOD and catalase activities in aortic segments. It is unlikely in these conditions that a fish oil-rich diet increases peroxynitrite generation at the vascular level that will impair ·NO effects. Furthermore, additional work is needed to elucidate the role of dietary fish oil and ·NO on low-density lipoprotein oxidation.

In conclusion, our study has demonstrated that a fish oil-rich diet upregulates eNOS expression. This, in turn, stimulates the accumulation of cGMP on vascular smooth muscle cells. The activation of the ·NO-cGMP pathway by fish oil represents a key physiological mechanism for the dynamic regulation of ·NO-mediated responses in the vasculature that is important to prevent arterial diseases.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Comision Interministerial de Ciencia y Tecnologia PM 98-0182 and Generalitat de Catalunya 1999SGR00266, and we thank Universitat de Barcelona for support to D. López.


   ACKNOWLEDGMENTS
 
We thank N. Clos and P. Fernandez from Serveis Científico-Tècnics of the Universitat de Barcelona for technical assistance in ESR evaluation and amino acid analysis, respectively, and Robin Rycroft for valuable assistance in the preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Mitjavila, Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avgda. Diagonal 645, E-08028 Barcelona, Spain (E-mail: mmitjavila{at}ub.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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